Dries Cardoen

Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers.

Genome‐wide analysis of alternative reproductive phenotypes in honeybee workers

A defining feature of social insects is the reproductive division of labour, in which workers usually forego all reproduction to help their mother queen to reproduce. However, little is known about the molecular basis of this spectacular form of altruism. Here, we compared gene expression patterns between nonreproductive, altruistic workers and reproductive, non‐altruistic workers in queenless honeybee colonies using a whole‐genome microarray analysis. Our results demonstrate massive differences in gene expression patterns between these two sets of workers, with a total of 1292 genes being differentially expressed. In nonreproductive workers, genes associated with energy metabolism and respiration, flight and foraging behaviour, detection of visible light, flight and heart muscle contraction and synaptic transmission were overexpressed relative to reproductive workers. This implies they probably had a higher whole‐body energy metabolism and activity rate and were most likely actively foraging, whereas same‐aged reproductive workers were not. This pattern is predicted from evolutionary theory, given that reproductive workers should be less willing to compromise their reproductive futures by carrying out high‐risk tasks such as foraging or other energetically expensive tasks. By contrast, reproductive workers mainly overexpressed oogenesis‐related genes compared to nonreproductive ones. With respect to key switches for ovary activation, several genes involved in steroid biosynthesis were upregulated in reproductive workers, as well as genes known to respond to queen and brood pheromones, genes involved in TOR and insulin signalling pathways and genes located within quantitative trait loci associated with reproductive capacity in honeybees. Overall, our results provide unique insight into the molecular mechanisms underlying alternative reproductive phenotypes in honeybee workers.

Mass spectrometric profiling of (neuro)-peptides in the worker honeybee, Apis mellifera.

The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography‐mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5‐like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5‐like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4‐based cross‐reactivity was detected between the honey bee Ag5‐like protein and its yellow jacket paralogue Ves v 5.

Worker honeybee sterility: a proteomic analysis of suppressed ovary activation.

Eusocial behavior is extensively studied in the honeybee, Apis mellifera, as it displays an extreme form of altruism. Honeybee workers are generally obligatory sterile in a bee colony headed by a queen, but the inhibition of ovary activation is lifted upon the absence of queen and larvae. Worker bees are then able to develop mature, viable eggs. The detailed repressive physiological mechanisms that are responsible for this remarkable phenomenon are as of yet largely unknown. Physiological studies today mainly focus on the transcriptome, while the proteome stays rather unexplored. Here, we present a quantitative 2-dimensional differential gel electrophoresis comparison between activated and inactivated worker ovaries and brains of reproductive and sterile worker bees, including a spot map of ovaries, containing 197 identified spots. Our findings suggest that suppression of ovary activation might involve a constant interplay between primordial oogenesis and subsequent degradation, which is probably mediated through steroid and neuropeptide hormone signaling. Additionally, the observation of higher viral protein loads in both the brains and ovaries of sterile workers is particularly noteworthy. This data set will be of great value for future research unraveling the physiological mechanisms underlying the altruistic sterility in honeybee workers.

Early changes in the pupal transcriptome of the flesh fly Sarcophagha crassipalpis to parasitization by the ectoparasitic wasp, Nasonia vitripennis

We investigated changes in the pupal transcriptome of the flesh fly Sarcophaga crassipalpis, 3 and 25 h after parasitization by the ectoparasitoid wasp, Nasonia vitripennis. These time points are prior to hatching of the wasp eggs, thus the results document host responses to venom injection, rather than feeding by the wasp larvae. Only a single gene appeared to be differentially expressed 3 h after parasitization. However, by 25 h, 128 genes were differentially expressed and expression patterns of a subsample of these genes were verified using RT-qPCR. Among the responsive genes were clusters of genes that altered the flys metabolism, development, induced immune responses, elicited detoxification responses, and promoted programmed cell death. Envenomation thus clearly alters the metabolic landscape and developmental fate of the fly host prior to subsequent penetration of the pupal cuticle by the wasp larva. Overall, this study provides new insights into the specific action of ectoparasitoid venoms.

Fast and Reliable Quantitative Peptidomics with labelpepmatch

The use of stable isotope tags in quantitative peptidomics offers many advantages, but the laborious identification of matching sets of labeled peptide peaks is still a major bottleneck. Here we present labelpepmatch, an R-package for fast and straightforward analysis of LC-MS spectra of labeled peptides. This open-source tool offers fast and accurate identification of peak pairs alongside an appropriate framework for statistical inference on quantitative peptidomics data, based on techniques from other -omics disciplines. A relevant case study on the desert locust Schistocerca gregaria proves our pipeline to be a reliable tool for quick but thorough explorative analyses.

A proteomic approach to neuropeptide function elucidation

Many of the diverse functions of neuropeptides are still elusive. As they are ideally suited to modulate traditional signaling, their added actions are not always detectable under standard laboratory conditions. The search for function assignment to peptide encoding genes can therefore greatly benefit from molecular information. Specific molecular changes resulting from neuropeptide signaling may direct researchers to yet unknown processes or conditions, for which studying these signaling systems may eventually lead to phenotypic confirmation. Here, we applied gel-based proteomics after pdf-1 neuropeptide gene knockout in the model organism Caenorhabditis elegans. It has previously been described that pdf-1 null mutants display a locomotion defect, being slower and making more turns and reversals than wild type worms. The vertebrate functional homolog of PDF-1, vasocative intestinal peptide (VIP), is known to influence a plethora of processes, which have so far not been investigated for pdf-1. Because proteins represent the actual effectors inside an organism, proteomic analysis can guide our view to novel pdf-1 actions in the nematode worm. Our data show that knocking out pdf-1 results in alteration of levels of proteins involved in fat metabolism, stress resistance and development. This indicates a possible conservation of VIP-like actions for pdf-1 in C. elegans.

Social evolution: when promiscuity breeds cooperation.

In social evolution theory, it has become common wisdom that close family ties should promote cooperative behaviour. Yet, in social insects, evidence is accumulating that queen promiscuity and low relatedness sometimes work better.

Life-prolonging measures for a dead theory?

In a recent review article, Selman and colleagues (Trends Ecol Evol 27:570–577, 2012) discuss the status quo of the oxidative stress theory of aging (OSTA) and how it links to life history evolution. They suggest that the OSTA should be tested in wild populations which might show effects masked in laboratory settings. We disagree with their propositions for several reasons. We argue that there is increasing evidence that reactive oxygen species (ROS) are not causally linked with aging and that ROS do not play a straightforward role in shaping life history evolution. We propose that laboratory animals and semi-wild populations rather than wild animals are suited best to test any hypothesized effect of reactive oxygen species. This is because data from controlled manipulative experiments rather than observational correlations are preferred to solve this issue. In addition, nonconventional model organisms will be useful in answering the question how relevant the OSTA could be for life history evolution.